N-Acetylglucosamine-1-P Uridylyltransferase 1 and 2 Are Required for Gametogenesis and Embryo Development in Arabidopsis thaliana 1
Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 3 Present address: Department of Horticulture, National Chung Hsing University, Taichung, Taiwan. 2
*Corresponding author: E-mail, [email protected]; Fax, +886-2-2782-7954. (Received May 5, 2014; Accepted September 6, 2014)
Keywords: Embryogenesis Gametogenesis Hexosamine biosynthetic pathway N-acetylglucosamine-1-P uridylyltransferase Sporophyte UDP-N-acetylglucosamine. Abbreviations: AGP, arabinogalactan protein; DAPI, 40 ,6-diamidino-2-phenylindole; DIC, differential interference contrast; dsRNAi, double-stranded RNA interference; FDA, fluorescein
diacetate; FG, female gametophyte; GlcNAc, N-acetylglucosamine; GlcNAc1pUT or GlcNA.UT, N-acetylglucosamine-1-P uridylyltransferase; GNA, glucosamine-6-P N-acetyltransferase; GPI, glycosylphosphatidylinositol; GUS, b-glucuronidase; HBP, hexosamine biosynthetic pathway; PMI, pollen mitosis I; qRTPCR, quantitative real-time PCR; RT–PCR, reverse transcription–PCR; SEM, scanning electron microscopy; TEM, transmission electron microscopy; Suc, sucrose; UDP-GlcNAc, UDP-N-acetylglucosamine.
Introduction N-Acetylglucosamine (GlcNAc) is the fundamental amino sugar residue in initiating N-glycan biosynthesis on the cytosolic side of the endoplasmic reticulum (Stanley et al. 2009). Furthermore, many cytosolic and nuclear proteins are O-glycosylated on Ser/Thr with a single GlcNAc residue; this type of modification is reversible and plays a role in protein stability, subcellular localization and protein–protein interactions (Hanover 2001, Wells and Hart 2003). GlcNAc also serves as a sugar moiety in glycolipids (Raetz and Whitfield 2002), glycosylphosphatidylinositol (GPI) anchor-linked protein synthesis (Hancock, 2004) and the yeast cell wall (chitin) (Carlos and Maia 1994). UDP-GlcNAc, an active form of GlcNAc, is synthesized through the so-called hexosamine biosynthetic pathway (HBP) and is conserved across eukaryotes. The first step in this pathway is the transamination of fructose-6-phosphate (Fru-6P) and glutamine into glucosamine-6-P (GlcN-6-P) and glutamate via a glutamine:Fru-6-P amidotransferase (Hassid et al. 1959). GlcN-6-P is subsequently N-acetylated by GlcN-6-P Nacetylase to N-acetylglucosamine-6-P (GlcNAc-6-P) followed by the conversion into GlcNAc-1-P via phospho-GlcNAc mutase. The final step is the formation of UDP-GlcNAc and PPi from the conversion of GlcNAc-1-P and UTP (Durand et al. 2008) catalyzed by N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) (Yang et al. 2010). GlcNAc1pUT in some organisms, such as yeast, Drosophila and humans, is encoded by one gene. A null mutation of yeast UAP1 (or QRI1) is lethal (Mio et al. 1998). The mutants of Drosophila UAP exhibit defects in dorsal closure and nervous system development (Schimmelpfeng et al. 2006). Although
Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127, Advance Access publication on 16 September 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
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Although N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) that catalyzes the final step of the hexosamine biosynthetic pathway and is conserved among, organisms, produces UDP-N-acetylglucosamine (UDP-GlcNAc), an essential sugar moiety involved in protein glycosylation and structural polymers, its biological function in plants remains unknown. In this study, two GlcNA.UT genes were characterized in Arabidopsis thaliana. The single mutants glcna.ut1 and glcna.ut2 revealed no obvious phenotype, but their homozygous double mutant was lethal, reflecting the functional redundancy of these genes in being essential for plant growth. Mutant plants, GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2, obtained from an F2-segregating population following reciprocal crosses of glcna.ut1 with glcna.ut2, displayed shorter siliques and fewer seed sets combined with impaired pollen viability and unfertilized ovules. Genetic analyses further demonstrated that the progeny of the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants, but not those of the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants, suffer from the aberrant transmission of (glcna.ut1 glcna.ut2) gametes. In parallel, cell biology analyses revealed a substantial defect in male gametophytes appearing during the late vacuolated or pollen mitosis I stages and that the female gametophyte is arrested during the uninucleate embryo sac stage in GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. Nevertheless, although the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited a normal transmission of (glcna.ut1 glcna.ut2) gametes and gametophytic development, the development of numerous embryos was arrested during the early globular stage within the embryo sacs. Collectively, despite having overlapping functions, the GlcNA.UT genes play an indispensable role in the unique mediation of gametogenesis and embryogenesis.
Regular Paper
Ya-Huei Chen1,2, Hwei-Ling Shen2, Pei-Jung Hsu2, San-Gwang Hwang2,3 and Wan-Hsing Cheng1,2,*
Y.-H. Chen et al. | GlcNAc1pUTs function in gametogenesis and embryogenesis
Results Isolation and characterization of the glcna.ut1 and glcna.ut2 mutants Two GlcNAc1pUTs, encoded by GlcNA.UT1 (At1g31070) and GlcNA.UT2 (At2g35020), were identified in Arabidopsis (Kotake et al. 2004, Yang et al. 2010, Kleczkowski et al. 2011). Both GlcNA.UT1 and GlcNA.UT2 contain 15 exons and 14 introns (Fig. 1A). The full-length cDNAs of GlcNA.UT1 and GlcNA.UT2, respectively, are 1,812 and 1,830 bp long (Fig. 1A) and encode deduced polypeptides with 505 and 502 amino acids. The GlcNAc1pUT1 and GlcNAc1pUT2 proteins share 1978
Fig. 1 Gene structure and expression patterns of the glcna.ut mutants. (A) Schematic diagrams of GlcNA.UT DNA and RNA transcript structures. The red arrows indicate the T-DNA insertion sites. (B) RT– PCR showing transcripts of the wild type (Col-0) and glcna.ut mutants. The seedlings that were grown on 1% Suc agar medium for 8 d were used for the RNA extraction and RT–PCR.
86% amino acid identity and contain two putative motifs for nucleotide binding (NB) and uridine binding (UB) (Yang et al. 2010). To better understand the molecular basis and biological role of GlcNA.UT genes, a reverse genetic approach was performed. We requested T-DNA-inserted mutants (Alonso et al. 2003) from the Arabidopsis Biological Research Center (ABRC). Two mutant lines were obtained from each GlcNA.UT gene. The glcna.ut1-1 (SALK_068977) and glcna.ut1-2 (SALK_015841) mutants had a T-DNA insertion at exon 3 and intron 7, respectively (Fig. 1A, arrows in red). Reverse transcription–PCR (RT–PCR) further demonstrated that these two mutants exhibited undetectable levels of the GlcNA.UT1 transcript but near normal levels of the GlcNA.UT2 transcript (Fig. 1B), suggesting that both of these mutants are knockout mutants. Similarly, two mutants, glcna.ut2-1 (SALK_150383) and glcna.ut2-2 (SALK_090658), were isolated and had a T-DNA insertion at intron 10 and exon 14, respectively (Fig. 1A, arrows in red). The glcna.ut2-1 and glcna.ut2-2 mutants showed no GlcNA.UT2 transcript but near normal levels of the GlcNA.UT1 transcript (Fig. 1B). However, a trace level of the short truncated transcript was conclusively observed in glcna.ut2-1 in the overexposed gel images (Supplementary Fig. S1A). Further cloning and sequencing revealed that this truncated transcript had a 78 bp deletion (77 bp in exon 10 and 1 bp in exon 11) and lacked amino acids 284–309. Thus, glcna.ut2-1 contained a truncated transcript, and glcna.ut2-2 was a knockout mutant. A phenotypic comparison indicated that no obvious phenotype was observed between the glcna.ut1 and glcna.ut2 single
Downloaded from http://pcp.oxfordjournals.org/ at Michigan State University on January 27, 2015
humans have one AGX gene, this gene generates two alternative splice forms, AGX1 and AGX2, which differ by a 17 residue peptide (Wang-Gillam et al. 1998) and confer the ability for oligomeric assembly (Peneff et al. 2001). Two UAPs in the red flour beetle (Tribolium castaneum) are important for development and survival but they differ in the regulation of the synthesis of cuticular or peritrophic matrix chitin (Arakane et al. 2011). In mammals, high levels of glucose may increase glycolytic flux and stimulate the HBP. Increased HBP metabolites have been linked to insulin resistance in mammalian cells (Marshall et al. 1991, McClain 2002). However, studies of the role of the HBP in plants are still limited. In Arabidopsis, the exogenous application of GlcN reduces hypocotyl elongation and the accumulation of reactive oxygen species through a hexokinase-mediated pathway (Ju et al. 2009). Moreover, the mutation of glucosamine-6-P N-acetyltransferase (GNA) in Arabidopsis leads to growth defects at high temperatures (28 C), the accumulation of lignin, the reduction of UDPGlcNAc and the interruption of N-glycosylation (Nozaki et al. 2012). In rice, the gna mutant exhibits a temperature-sensitive short-root phenotype and defects in glycosylated proteins (Jiang et al. 2005). Although two Arabidopsis GlcNAc1pUTs have been identified and show multiple substrate specificity (Yang et al. 2010), their biological function remains unknown. In this study, the biological functions of these two Arabidopsis GlcNAc1pUTs, encoded by GlcNA.UT genes, were characterized through molecular, genetic and cellular approaches. The GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants and iU1 double-stranded RNA interference (dsRNAi) lines exhibited defects in pollen and silique development. In agreement with these phenotypes, cellular analyses indicated that defects in the aberrant transmission of (glcna.ut1 glcna.ut2) gametes and in both male and female gametophytic development occur in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. Nevertheless, despite normal gamete transmission and gametophytic development, the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited defects in numerous embryo development events during the early globular stage in the embryo sacs. Collectively, these two GlcNA.UT genes play essential roles in normal plant growth and development through the unique control of gametogenesis and embryogenesis and are most probably associated with the sporophytic effect.
Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127
mutants and the wild type (Fig. 2A). To investigate whether GlcNA.UT1 and GlcNA.UT2 share functional redundancy, we attempted to identify the glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutant from an F2-segregating population that was derived from the crosses of glcna.ut1-1 with glcna.ut2-1 and glcna.ut1-2 with glcna.ut2-2. After genotyping 280 seedlings of the glcna.ut1-1glcna.ut2-1 F2 progeny, we were unable to identify any homozygous double mutants. These data suggest that the double mutant is most probably lethal and that the GlcNA.UT1 and GlcNA.UT2 genes share redundant functions that are essential for plant growth and development.
During the failure to isolate the glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutants in the F2 segregating population that was derived from the reciprocal crosses of glcna.ut1 and glcna.ut2, a short silique phenotype was observed in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. However, similar to the glcna.ut1 and glcna.ut2 single mutants, the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 plants resembled the wild type with respect to plant size and silique length (Fig. 2A–C). These data indicate that in the single mutant glcna.ut1 background, one copy of the wild-type GlcNA.UT2 allele is sufficient to maintain normal silique growth, whereas in the single mutant glcna.ut2 background, one copy of the GlcNA.UT1 allele is not adequate for normal silique growth. Consistent with the short siliques that were observed in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants, the total seed number per silique in these mutants was also greatly reduced (Fig. 2D). The glcna.ut2-2 mutants exhibited a slight reduction in the seed number per silique but to a much lesser extent compared with the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants. Thus, these data suggest that the short silique length is generally associated with a reduction in the total seed number. An analysis of the siliques of the GlcNA.UT1/glcna.ut1-1 glcna.ut21/glcna.ut2-1 plants demonstrated the presence of numerous unoccupied spaces, in which the ovules were small and white (Fig. 3A vs. B, arrowheads in red), suggesting that these ovules were probably unfertilized. In contrast, very few vacancies in the siliques of glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 (Fig. 3C, D) and Col-0 (Fig. 3A) plants were observed. Nevertheless, some seeds of the glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 plants were pale green or yellow (Fig. 3C, arrowheads in white) and were slightly smaller in size than the normal developing seeds in the same silique or the wild type. These discolored seeds probably turned brown and shrunk (Fig. 3D, E) because we also observed brown and shrunk seeds together with the wild-type-like seeds in the later stage within the same silique. Notably, the percentage of the shrunken seeds per silique was 22% in the glcna.ut1-1/glcna.ut11 GlcNA.UT2/glcna.ut2-1 siliques and 23% in the glcna.ut1-2/ glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 siliques (Fig. 3F). These
Genetic analysis reveals the unequal transmission of the glcna.ut gametes To investigate further the genetic relevance of GlcNA.UT1 and GlcNA.UT2, an analysis of segregation among the progeny of the self-pollinated glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 and GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 plants was conducted. Consistent with the above-mentioned results, not only the glcna.ut1/ glcna.ut1 GlcNA.UT2/glcna.ut2 but also the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 progeny population produced no glcna.ut1/glcna.ut1 glcna.ut2/glcna.ut2 double mutants in a total of 502 plants of self-pollinated populations (Table 1), again suggesting that the function of GlcNA.UT is essential for plant survival in Arabidopsis. Based on the absence of glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutants in the population, the remnant genotypes of the progeny that were derived from selfed GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 or glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 were analyzed. The glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 plants will presumably generate two types of gametes, i.e. (glcna.ut1 GlcNA.UT2) and (glcna.ut1 glcna.ut2), and, after self-pollination, will lead to two types of progeny, i.e. glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/GlcNA.UT2 and glcna.ut1-1/glcna.ut1-1 GlcNA. UT2/glcna.ut2-1, at expected ratios of 33% and 67%, respectively. As expected, a 66% segregation rate of identical parental genotype from the selfed glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/ glcna.ut2-1 occurred in the next segregating population (Table 1). Similarly, 61% of the progeny of the self-pollinated glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 plants produced genotypes that were identical to those of their parents. These observed values (66% and 61%) are very close to the expected 67%. Therefore, these data suggest that the transmission of the (glcna.ut1 glcna.ut2) gamete was unaffected in the self-pollinated glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 plants. However, in the progeny of the self-pollinated GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1 plants, only 18% of the offspring had the same parental genotype, and an even lower ratio (8%) of parental genotypes was identified from the progeny of the GlcNA.UT1/glcna.ut1-2 glcna.ut2-2/glcna.ut2-2 plants (Table 1). These results suggest that a reduction of (glcna.ut1 glcna.ut2) co-transmission occurs in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 plants (Table 1). Furthermore, the distorted segregation rates in the selfed GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 progeny but not in the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 progeny indicate that the GlcNA.UT2 and GlcNA.UT1 alleles may contribute unequally to gametophytic development. Taken together, these results indicate that although GlcNA.UT1 and GlcNA.UT2 act redundantly in
Downloaded from http://pcp.oxfordjournals.org/ at Michigan State University on January 27, 2015
GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 and glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutants exhibit differences in silique length and seed number
values are coincidently close to a quarter of the segregation ratio from their self-pollinated F1 population, which, however, were considerably low in the GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 mutants (Fig. 3F). The shrunken seeds in glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 plants showed no germination (0%) compared with the wild-type-like seeds (98.4%) that were grown on the same individual plants (Fig. 3G).
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Y.-H. Chen et al. | GlcNAc1pUTs function in gametogenesis and embryogenesis
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Fig. 2 Defects in silique length and seed development in single or double glcna.ut1 and glcna.ut2 allelic mutants. (A) Comparison of the plant phenotype among the genotypes. The plants were grown in soil for 38 d. (B–D) Comparison of the silique length and seed number per silique. The plants were grown in soil for 45 d. The siliques at the fifth to tenth positions of the primary flower shoots were removed (B), and the average silique length was measured (C). The values in (C) are the mean ± SD of three independent experiments, each with n = 18. **P < 0.01, Student’s t-test. After removal of Chl by ethanol, the average seed number per silique was calculated from the siliques at the fifth to eighth positions of the primary flower shoots (D) that were derived from (B). The values in (D) are the mean ± SD of three independent experiments, each with n = 12. **P < 0.01, Student’s t-test.
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Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127
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Fig. 3 Degeneration of seed development in the siliques of the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutants. (A and B) Developing seeds on greening siliques were derived from the wild-type (A) or from the GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1 (B) plants. The plants were grown in soil for 38 d. The arrowheads in red indicate the unfertilized ovules. (C–E) The various developmental stages of the seeds in the glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 mutant siliques. The plants were grown in soil for 38 d (C and D) or 57 d (E). The shrunken or discolored seeds are indicated by white arrowheads. (F) Yellow siliques at the third to eighth positions of the primary flower shoots on the 57day-old plants were harvested, and the ratio of shrunken to normal seeds was calculated using a dissection microscope. The values are the mean ± SD of two independent experiments, each with siliques (n = 8) from two plants. **P < 0.01, Student’s t-test. Scale bar = 1 mm. (G) Germination of wild-type-like and shrunken seeds of glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2. Seeds were grown on medium for 1 week. The values are the mean ± SD of three independent experiments, each with 42–52 seeds. Scale bar = 1 cm.
Table 1 Self-pollination of GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 reveals a defect in gamete transmission Parent genotypea
Expected genotypeb (parent genotype) in F1
Observed genotype (parent genotype) in F1
2 (P-value)c
GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1
(selfed)
96/144 (66%)
26/144 (18%)
153.125 (
Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 3 Present address: Department of Horticulture, National Chung Hsing University, Taichung, Taiwan. 2
*Corresponding author: E-mail, [email protected]; Fax, +886-2-2782-7954. (Received May 5, 2014; Accepted September 6, 2014)
Keywords: Embryogenesis Gametogenesis Hexosamine biosynthetic pathway N-acetylglucosamine-1-P uridylyltransferase Sporophyte UDP-N-acetylglucosamine. Abbreviations: AGP, arabinogalactan protein; DAPI, 40 ,6-diamidino-2-phenylindole; DIC, differential interference contrast; dsRNAi, double-stranded RNA interference; FDA, fluorescein
diacetate; FG, female gametophyte; GlcNAc, N-acetylglucosamine; GlcNAc1pUT or GlcNA.UT, N-acetylglucosamine-1-P uridylyltransferase; GNA, glucosamine-6-P N-acetyltransferase; GPI, glycosylphosphatidylinositol; GUS, b-glucuronidase; HBP, hexosamine biosynthetic pathway; PMI, pollen mitosis I; qRTPCR, quantitative real-time PCR; RT–PCR, reverse transcription–PCR; SEM, scanning electron microscopy; TEM, transmission electron microscopy; Suc, sucrose; UDP-GlcNAc, UDP-N-acetylglucosamine.
Introduction N-Acetylglucosamine (GlcNAc) is the fundamental amino sugar residue in initiating N-glycan biosynthesis on the cytosolic side of the endoplasmic reticulum (Stanley et al. 2009). Furthermore, many cytosolic and nuclear proteins are O-glycosylated on Ser/Thr with a single GlcNAc residue; this type of modification is reversible and plays a role in protein stability, subcellular localization and protein–protein interactions (Hanover 2001, Wells and Hart 2003). GlcNAc also serves as a sugar moiety in glycolipids (Raetz and Whitfield 2002), glycosylphosphatidylinositol (GPI) anchor-linked protein synthesis (Hancock, 2004) and the yeast cell wall (chitin) (Carlos and Maia 1994). UDP-GlcNAc, an active form of GlcNAc, is synthesized through the so-called hexosamine biosynthetic pathway (HBP) and is conserved across eukaryotes. The first step in this pathway is the transamination of fructose-6-phosphate (Fru-6P) and glutamine into glucosamine-6-P (GlcN-6-P) and glutamate via a glutamine:Fru-6-P amidotransferase (Hassid et al. 1959). GlcN-6-P is subsequently N-acetylated by GlcN-6-P Nacetylase to N-acetylglucosamine-6-P (GlcNAc-6-P) followed by the conversion into GlcNAc-1-P via phospho-GlcNAc mutase. The final step is the formation of UDP-GlcNAc and PPi from the conversion of GlcNAc-1-P and UTP (Durand et al. 2008) catalyzed by N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) (Yang et al. 2010). GlcNAc1pUT in some organisms, such as yeast, Drosophila and humans, is encoded by one gene. A null mutation of yeast UAP1 (or QRI1) is lethal (Mio et al. 1998). The mutants of Drosophila UAP exhibit defects in dorsal closure and nervous system development (Schimmelpfeng et al. 2006). Although
Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127, Advance Access publication on 16 September 2014, available online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]
Downloaded from http://pcp.oxfordjournals.org/ at Michigan State University on January 27, 2015
Although N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT) that catalyzes the final step of the hexosamine biosynthetic pathway and is conserved among, organisms, produces UDP-N-acetylglucosamine (UDP-GlcNAc), an essential sugar moiety involved in protein glycosylation and structural polymers, its biological function in plants remains unknown. In this study, two GlcNA.UT genes were characterized in Arabidopsis thaliana. The single mutants glcna.ut1 and glcna.ut2 revealed no obvious phenotype, but their homozygous double mutant was lethal, reflecting the functional redundancy of these genes in being essential for plant growth. Mutant plants, GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2, obtained from an F2-segregating population following reciprocal crosses of glcna.ut1 with glcna.ut2, displayed shorter siliques and fewer seed sets combined with impaired pollen viability and unfertilized ovules. Genetic analyses further demonstrated that the progeny of the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants, but not those of the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants, suffer from the aberrant transmission of (glcna.ut1 glcna.ut2) gametes. In parallel, cell biology analyses revealed a substantial defect in male gametophytes appearing during the late vacuolated or pollen mitosis I stages and that the female gametophyte is arrested during the uninucleate embryo sac stage in GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. Nevertheless, although the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited a normal transmission of (glcna.ut1 glcna.ut2) gametes and gametophytic development, the development of numerous embryos was arrested during the early globular stage within the embryo sacs. Collectively, despite having overlapping functions, the GlcNA.UT genes play an indispensable role in the unique mediation of gametogenesis and embryogenesis.
Regular Paper
Ya-Huei Chen1,2, Hwei-Ling Shen2, Pei-Jung Hsu2, San-Gwang Hwang2,3 and Wan-Hsing Cheng1,2,*
Y.-H. Chen et al. | GlcNAc1pUTs function in gametogenesis and embryogenesis
Results Isolation and characterization of the glcna.ut1 and glcna.ut2 mutants Two GlcNAc1pUTs, encoded by GlcNA.UT1 (At1g31070) and GlcNA.UT2 (At2g35020), were identified in Arabidopsis (Kotake et al. 2004, Yang et al. 2010, Kleczkowski et al. 2011). Both GlcNA.UT1 and GlcNA.UT2 contain 15 exons and 14 introns (Fig. 1A). The full-length cDNAs of GlcNA.UT1 and GlcNA.UT2, respectively, are 1,812 and 1,830 bp long (Fig. 1A) and encode deduced polypeptides with 505 and 502 amino acids. The GlcNAc1pUT1 and GlcNAc1pUT2 proteins share 1978
Fig. 1 Gene structure and expression patterns of the glcna.ut mutants. (A) Schematic diagrams of GlcNA.UT DNA and RNA transcript structures. The red arrows indicate the T-DNA insertion sites. (B) RT– PCR showing transcripts of the wild type (Col-0) and glcna.ut mutants. The seedlings that were grown on 1% Suc agar medium for 8 d were used for the RNA extraction and RT–PCR.
86% amino acid identity and contain two putative motifs for nucleotide binding (NB) and uridine binding (UB) (Yang et al. 2010). To better understand the molecular basis and biological role of GlcNA.UT genes, a reverse genetic approach was performed. We requested T-DNA-inserted mutants (Alonso et al. 2003) from the Arabidopsis Biological Research Center (ABRC). Two mutant lines were obtained from each GlcNA.UT gene. The glcna.ut1-1 (SALK_068977) and glcna.ut1-2 (SALK_015841) mutants had a T-DNA insertion at exon 3 and intron 7, respectively (Fig. 1A, arrows in red). Reverse transcription–PCR (RT–PCR) further demonstrated that these two mutants exhibited undetectable levels of the GlcNA.UT1 transcript but near normal levels of the GlcNA.UT2 transcript (Fig. 1B), suggesting that both of these mutants are knockout mutants. Similarly, two mutants, glcna.ut2-1 (SALK_150383) and glcna.ut2-2 (SALK_090658), were isolated and had a T-DNA insertion at intron 10 and exon 14, respectively (Fig. 1A, arrows in red). The glcna.ut2-1 and glcna.ut2-2 mutants showed no GlcNA.UT2 transcript but near normal levels of the GlcNA.UT1 transcript (Fig. 1B). However, a trace level of the short truncated transcript was conclusively observed in glcna.ut2-1 in the overexposed gel images (Supplementary Fig. S1A). Further cloning and sequencing revealed that this truncated transcript had a 78 bp deletion (77 bp in exon 10 and 1 bp in exon 11) and lacked amino acids 284–309. Thus, glcna.ut2-1 contained a truncated transcript, and glcna.ut2-2 was a knockout mutant. A phenotypic comparison indicated that no obvious phenotype was observed between the glcna.ut1 and glcna.ut2 single
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humans have one AGX gene, this gene generates two alternative splice forms, AGX1 and AGX2, which differ by a 17 residue peptide (Wang-Gillam et al. 1998) and confer the ability for oligomeric assembly (Peneff et al. 2001). Two UAPs in the red flour beetle (Tribolium castaneum) are important for development and survival but they differ in the regulation of the synthesis of cuticular or peritrophic matrix chitin (Arakane et al. 2011). In mammals, high levels of glucose may increase glycolytic flux and stimulate the HBP. Increased HBP metabolites have been linked to insulin resistance in mammalian cells (Marshall et al. 1991, McClain 2002). However, studies of the role of the HBP in plants are still limited. In Arabidopsis, the exogenous application of GlcN reduces hypocotyl elongation and the accumulation of reactive oxygen species through a hexokinase-mediated pathway (Ju et al. 2009). Moreover, the mutation of glucosamine-6-P N-acetyltransferase (GNA) in Arabidopsis leads to growth defects at high temperatures (28 C), the accumulation of lignin, the reduction of UDPGlcNAc and the interruption of N-glycosylation (Nozaki et al. 2012). In rice, the gna mutant exhibits a temperature-sensitive short-root phenotype and defects in glycosylated proteins (Jiang et al. 2005). Although two Arabidopsis GlcNAc1pUTs have been identified and show multiple substrate specificity (Yang et al. 2010), their biological function remains unknown. In this study, the biological functions of these two Arabidopsis GlcNAc1pUTs, encoded by GlcNA.UT genes, were characterized through molecular, genetic and cellular approaches. The GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants and iU1 double-stranded RNA interference (dsRNAi) lines exhibited defects in pollen and silique development. In agreement with these phenotypes, cellular analyses indicated that defects in the aberrant transmission of (glcna.ut1 glcna.ut2) gametes and in both male and female gametophytic development occur in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. Nevertheless, despite normal gamete transmission and gametophytic development, the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited defects in numerous embryo development events during the early globular stage in the embryo sacs. Collectively, these two GlcNA.UT genes play essential roles in normal plant growth and development through the unique control of gametogenesis and embryogenesis and are most probably associated with the sporophytic effect.
Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127
mutants and the wild type (Fig. 2A). To investigate whether GlcNA.UT1 and GlcNA.UT2 share functional redundancy, we attempted to identify the glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutant from an F2-segregating population that was derived from the crosses of glcna.ut1-1 with glcna.ut2-1 and glcna.ut1-2 with glcna.ut2-2. After genotyping 280 seedlings of the glcna.ut1-1glcna.ut2-1 F2 progeny, we were unable to identify any homozygous double mutants. These data suggest that the double mutant is most probably lethal and that the GlcNA.UT1 and GlcNA.UT2 genes share redundant functions that are essential for plant growth and development.
During the failure to isolate the glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutants in the F2 segregating population that was derived from the reciprocal crosses of glcna.ut1 and glcna.ut2, a short silique phenotype was observed in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants. However, similar to the glcna.ut1 and glcna.ut2 single mutants, the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 plants resembled the wild type with respect to plant size and silique length (Fig. 2A–C). These data indicate that in the single mutant glcna.ut1 background, one copy of the wild-type GlcNA.UT2 allele is sufficient to maintain normal silique growth, whereas in the single mutant glcna.ut2 background, one copy of the GlcNA.UT1 allele is not adequate for normal silique growth. Consistent with the short siliques that were observed in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants, the total seed number per silique in these mutants was also greatly reduced (Fig. 2D). The glcna.ut2-2 mutants exhibited a slight reduction in the seed number per silique but to a much lesser extent compared with the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutants. Thus, these data suggest that the short silique length is generally associated with a reduction in the total seed number. An analysis of the siliques of the GlcNA.UT1/glcna.ut1-1 glcna.ut21/glcna.ut2-1 plants demonstrated the presence of numerous unoccupied spaces, in which the ovules were small and white (Fig. 3A vs. B, arrowheads in red), suggesting that these ovules were probably unfertilized. In contrast, very few vacancies in the siliques of glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 (Fig. 3C, D) and Col-0 (Fig. 3A) plants were observed. Nevertheless, some seeds of the glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 plants were pale green or yellow (Fig. 3C, arrowheads in white) and were slightly smaller in size than the normal developing seeds in the same silique or the wild type. These discolored seeds probably turned brown and shrunk (Fig. 3D, E) because we also observed brown and shrunk seeds together with the wild-type-like seeds in the later stage within the same silique. Notably, the percentage of the shrunken seeds per silique was 22% in the glcna.ut1-1/glcna.ut11 GlcNA.UT2/glcna.ut2-1 siliques and 23% in the glcna.ut1-2/ glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 siliques (Fig. 3F). These
Genetic analysis reveals the unequal transmission of the glcna.ut gametes To investigate further the genetic relevance of GlcNA.UT1 and GlcNA.UT2, an analysis of segregation among the progeny of the self-pollinated glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 and GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 plants was conducted. Consistent with the above-mentioned results, not only the glcna.ut1/ glcna.ut1 GlcNA.UT2/glcna.ut2 but also the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 progeny population produced no glcna.ut1/glcna.ut1 glcna.ut2/glcna.ut2 double mutants in a total of 502 plants of self-pollinated populations (Table 1), again suggesting that the function of GlcNA.UT is essential for plant survival in Arabidopsis. Based on the absence of glcna.ut1/glcna.ut1 glcna.ut2/ glcna.ut2 double mutants in the population, the remnant genotypes of the progeny that were derived from selfed GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 or glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 were analyzed. The glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 plants will presumably generate two types of gametes, i.e. (glcna.ut1 GlcNA.UT2) and (glcna.ut1 glcna.ut2), and, after self-pollination, will lead to two types of progeny, i.e. glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/GlcNA.UT2 and glcna.ut1-1/glcna.ut1-1 GlcNA. UT2/glcna.ut2-1, at expected ratios of 33% and 67%, respectively. As expected, a 66% segregation rate of identical parental genotype from the selfed glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/ glcna.ut2-1 occurred in the next segregating population (Table 1). Similarly, 61% of the progeny of the self-pollinated glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 plants produced genotypes that were identical to those of their parents. These observed values (66% and 61%) are very close to the expected 67%. Therefore, these data suggest that the transmission of the (glcna.ut1 glcna.ut2) gamete was unaffected in the self-pollinated glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 plants. However, in the progeny of the self-pollinated GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1 plants, only 18% of the offspring had the same parental genotype, and an even lower ratio (8%) of parental genotypes was identified from the progeny of the GlcNA.UT1/glcna.ut1-2 glcna.ut2-2/glcna.ut2-2 plants (Table 1). These results suggest that a reduction of (glcna.ut1 glcna.ut2) co-transmission occurs in the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 plants (Table 1). Furthermore, the distorted segregation rates in the selfed GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 progeny but not in the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 progeny indicate that the GlcNA.UT2 and GlcNA.UT1 alleles may contribute unequally to gametophytic development. Taken together, these results indicate that although GlcNA.UT1 and GlcNA.UT2 act redundantly in
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GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 and glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutants exhibit differences in silique length and seed number
values are coincidently close to a quarter of the segregation ratio from their self-pollinated F1 population, which, however, were considerably low in the GlcNA.UT1/glcna.ut1 glcna.ut2/ glcna.ut2 mutants (Fig. 3F). The shrunken seeds in glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2 plants showed no germination (0%) compared with the wild-type-like seeds (98.4%) that were grown on the same individual plants (Fig. 3G).
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Y.-H. Chen et al. | GlcNAc1pUTs function in gametogenesis and embryogenesis
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Fig. 2 Defects in silique length and seed development in single or double glcna.ut1 and glcna.ut2 allelic mutants. (A) Comparison of the plant phenotype among the genotypes. The plants were grown in soil for 38 d. (B–D) Comparison of the silique length and seed number per silique. The plants were grown in soil for 45 d. The siliques at the fifth to tenth positions of the primary flower shoots were removed (B), and the average silique length was measured (C). The values in (C) are the mean ± SD of three independent experiments, each with n = 18. **P < 0.01, Student’s t-test. After removal of Chl by ethanol, the average seed number per silique was calculated from the siliques at the fifth to eighth positions of the primary flower shoots (D) that were derived from (B). The values in (D) are the mean ± SD of three independent experiments, each with n = 12. **P < 0.01, Student’s t-test.
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Plant Cell Physiol. 55(11): 1977–1993 (2014) doi:10.1093/pcp/pcu127
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Fig. 3 Degeneration of seed development in the siliques of the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutants. (A and B) Developing seeds on greening siliques were derived from the wild-type (A) or from the GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1 (B) plants. The plants were grown in soil for 38 d. The arrowheads in red indicate the unfertilized ovules. (C–E) The various developmental stages of the seeds in the glcna.ut1-1/glcna.ut1-1 GlcNA.UT2/glcna.ut2-1 mutant siliques. The plants were grown in soil for 38 d (C and D) or 57 d (E). The shrunken or discolored seeds are indicated by white arrowheads. (F) Yellow siliques at the third to eighth positions of the primary flower shoots on the 57day-old plants were harvested, and the ratio of shrunken to normal seeds was calculated using a dissection microscope. The values are the mean ± SD of two independent experiments, each with siliques (n = 8) from two plants. **P < 0.01, Student’s t-test. Scale bar = 1 mm. (G) Germination of wild-type-like and shrunken seeds of glcna.ut1-2/glcna.ut1-2 GlcNA.UT2/glcna.ut2-2. Seeds were grown on medium for 1 week. The values are the mean ± SD of three independent experiments, each with 42–52 seeds. Scale bar = 1 cm.
Table 1 Self-pollination of GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 reveals a defect in gamete transmission Parent genotypea
Expected genotypeb (parent genotype) in F1
Observed genotype (parent genotype) in F1
2 (P-value)c
GlcNA.UT1/glcna.ut1-1 glcna.ut2-1/glcna.ut2-1
(selfed)
96/144 (66%)
26/144 (18%)
153.125 (
